Myosin light chain 3F regulatory sequences confer regionalized cardiac and skeletal muscle expression in transgenic mice.

Abstract

Abstract. The myosin light chain 1F/3F locus contains two independent promoters, MLC1F and MLC3F, which are differentially activated during skeletal muscle development. Transcription at this locus is regulated by a 3' skeletal muscle enhancer element, which directs correct temporal and tissuespecific expression from the MLC1F promoter in transgenic mice. To investigate the role of this enhancer in regulation of the MLC3F promoter in vivo, we have analyzed reporter gene expression in transgenic mice containing lacZ under transcriptional control of the mouse MLC3F promoter and 3' enhancer element. Our results show that these regulatory elements direct strong expression of lacZ in skeletal muscle; the transgene, however, is activated 4-5 d before the endogenous MLC3F promoter, at the time of initiation of MLC1F transcription. In adult mice, transgene activity is downregulated in muscles that have reduced contributions of type 1/13 fibers (soleus and diaphragm). The rostrocaudal positional gradient of transgene expression documented for MLC1F transgenic mice (Donoghue, M., J. P. Merlie, N. Rosenthai, and J. R. Sanes. 1991. Proc. Natl. Acad. Sci. USA. 88:5847-5851) is not seen in MLC3F transgenic mice. Although MLC3F was previously thought to be restricted to skeletal striated muscle, the MLC3F-lacZ transgene is expressed in cardiac muscle from 7.5 d of development in a spatially restricted manner in the atria and left ventricular compartments, suggesting that transcriptional differences exist between cardiomyocytes in left and right compartments of the heart. We show here that transgene-directed expression of the MLC3F promoter reflects low level expression of endogenous MLC3F transcripts in the mouse heart. URING striated muscle development, a dynamic and complex pattern of structural gene expression generates the diversity of muscle types found in the adult vertebrate. Regulation of the majority of sarcomeric genes is under transcriptional control mediated by several families of regulatory proteins, including those of the MyoD and MEF2 transcription factors (Weintraub, 1993;Yu et al., 1992). Through interactions with cis-acfing regulatory elements, these factors ensure the expression of specific genes whose products are required in particular subsets of cardiac and skeletal musculature. A large number of muscle-specific promoter and enhancer elements have been identified as important in tissue culture (see Rosenthal, 1989); dissection, however, of the complex spatial and temporal control of muscle-specific gene expression in vivo, which is not recapitulated in vitro, requires direct analysis of putative regulatory regions in transgenic mice. Transgenic studies have demonstrated the complexity of cis-acflng elements controlling the expression of particular muscle-specific genes in different striated muscle types, for example, the Address correspondence to M. Buckingham, CNRS URA 1947,

The homeobox gene Arx is a novel positive regulator of embryonic myogenesis

“…7,39 In Tg:3F-nlacZ-2E mice, b-galactosidase expression is restricted to differentiated skeletal and cardiac muscle. 37 Myf5 nlacZ/nlacZ mice have a loss of function of both Myf5 and Mrf4 at embryonic stages, whereas in Myf5 loxP/loxP Mrf4 expression appears unaffected. 7 Double mutant Myf5 loxP/loxP MyoD À/À and Myf5 nlacZ/nlacZ (Mrf4):MyoD À/À embryos were obtained by breeding double heterozygous mice.…”

“…The mutant and transgenic animals and genotyping primers used in this work were published: Pax3 GFP , 5 Arx nlacZ , 13 Tg: 3F-nlacZ-2E, 37 MyoD À/À , 38 Myf5 GFPÀP , Myf5 loxP , Myf5 nlacZ . 7,39 In Tg:3F-nlacZ-2E mice, b-galactosidase expression is restricted to differentiated skeletal and cardiac muscle.…”

Non-viral gene delivery in skeletal muscle: a protein factory

“…Of the non-viral promoters employed, some such as b actin have general activity within eukaryote cells. 30,31 Some promoters control expression in a cell-or tissue-specific manner eg a skeletal muscle actin promoter, 32 myosin light chain 3F promoter 33 and muscle creatine kinase (MCK) promoter 34 which are activated specifically in muscle. The best example of promoters of high cell specificity is the ventricle-specific myosin light chain-2 which is activated exclusively in cardiac myocytes.…”

“…It is well documented that plasmid DNA may persist within target cells with no detectable expression. 33 It is not well understood how transgene expression is regulated in muscle. Studies of transgene expression with viral infection systems suggest that there is room for improvement of transgene expression at both the transcriptional and post-transcriptional levels.…”

Morphology of the Lumbar Transversospinal Muscles Examined in a Mouse Bearing a Muscle Fiber‐Specific Nuclear Marker

“…To answer these questions, we used a transgenic mouse line (MLC3F-nlacZ; Kelly et al, 1995) that bears a clear marker in every muscle nucleus of the mature fibers. Visualization of the marker requires only a simple histochemical process that can be performed on intact tissues before dissection, thereby allowing clear and unambiguous demarcation of the boundaries between muscle fibers and other non-muscle elements such as ligaments, tendons, and aponeuroses.…”